To date our genetic and biochemical studies indicate that our original Hsp70 mutant impairs prions either by promoting self-association of amyloid fibers into large aggregates or by interfering with the disentangling of amyloid fibers from such higher-order aggregates. Unlike other in vitro systems used for studying interaction of chaperones with amyloid, the system we are developing allows testing the ability of chaperones or chaotropic agents to disentangle aggregated amyloid fibers. The basis of the system is a version of a multiply tagged yeast prion domain that can be differentially labeled by fluorescent and other moieties. Fibers are labeled by either or both probes, and those containing both can be labeled either sequentially or simultaneously. Fluorescence can be used in conjunction with physical methods such as fractionation and immunoblotting to test the extent to which chaperones bind to or break fibers. Our system will also allow monitoring the action of chaperones in the disentangling of polymers from aggregates. Ultimately, products generated from in vitro reactions will be used for infecting cells to test our predictions of how chaperone interaction with amyloid affects prion infectivity.[unreadable] In addition to studying effect of chaperones on amyloid formed from wild type prion protein, we are interested in studying how alterations in primary structure of prion proteins can affect chaperone interactions. To this end we have been isolating mutants of prion proteins that interfere with propagation of prions formed of wild type protein or that form prions with weakened phenotypes when the wild type protein is absent.